Candida albicans is the most common cause of human fungal infection. It is commonly found as a commensal organism on the mucosal membrane of healthy individuals, but can become infectious under conditions that compromise host defense. Oropharyngeal and esophageal candidiasis are the most prevalent mycotic infections among patients with AIDS (80-90%). More invasive systemic infections are associated with neutropenic patients. Several properties of C. albicans are associated with its ability to cause disease, most notably its capacity to switch between yeast and filamentous forms. Cytological observations have revealed that during the switch between yeast and hyphal growth the vacuole of C. albicans undergoes a rapid expansion to occupy the majority of parental and subapical cell compartments, while the cytoplasmic material migrates at the hyphal tip. We have previously reported that a C. albicans mutant defective in vacuole biogenesis is afilamentous. We hypothesize that vacuole expansion is an essential event during yeast-hyphal differentiation, and that this expansion is mediated through the fusion of membranous vesicles derived from other cellular organelles such as the pre-vacuole compartment and Golgi, with the vacuole. The objective of this proposal is to firstly confirm that vacuole expansion is a requirement for filament formation and secondly to determine which of the trafficking pathways to the vacuole mediate filament induced expansion. Specifically we will construct mutant strains defective in each delivery pathway to the vacuole and assess the affect on vacuole biogenesis and filamentation. We will also construct GFP fusion products to confirm the transport steps that are defective in our mutants. The long term research goal is to establish the role the vacuole plays in processes of adaptation and differentiation in C. albicans. These events are likely to be essential in this organisms' ability to survive within the host environment, and initiate infection. Thus, selected mutant strains will also be tested in the systemic mouse model. The information obtained in this study on the role of the fungal vacuole in pathogenesis may also aid in the design of new anti-fungal treatment strategies.